Adipose tissue centrifuge and method of use

ABSTRACT

A centrifuge device is provided for the sizing and separation of constituents of a biologic mixture, e.g., adipose tissue. The device provides for the mechanical breaking down of the fibrous structure in the tissue by centrifugation causing the tissue to pass through a mesh element, or a sizing helix, or an extrusion element, whereupon the material is reduced to a slurry. This processed material may then be separated by centrifugation into its constituents, in order to harvest the fraction containing the multipotent cells. These multipotent cells may be utilized for various medical procedures to stimulate healing and tissue regeneration.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/949,714, filed 10 Apr. 2018, now U.S. Pat. No. 10,711,239, which is adivisional application of U.S. application Ser. No. 14/610,613, filed 30Jan. 2015, now U.S. Pat. No. 10,125,345, which claims the benefit ofU.S. Provisional Application No. 61/934,069, filed 31 Jan. 2014, theentire contents of each of which are hereby incorporated by reference.

BACKGROUND

Multipotent cells are known to be useful in various medical proceduresto assist in the healing of an affected area of a patient, for exampleby providing enhanced cellular regeneration of a treatment site. Themultipotent cells can be sourced from various tissues of the body of aliving being for use in a surgical procedure. The multipotent cells maybe autologous, where the patient is the donor for the cells that areused to treat the same patient. The term “multipotent cells” includesadipose-derived stem cells, which have also been described asadipose-derived stem/stromal cells, adipose-derived adult stem cells,adipose-derived adult stromal cells, adipose-derived stromal cells,adipose stromal cells, adipose mesenchymal stem cells, lipoblast,pericyte, preadipocyte, and processed lipoaspirate cells.

It is well known that adipose tissue in the human body containssignificant numbers of multipotent cells, in fact, far more multipotentcells are stored per unit volume in fat than in bone marrow. Someestimates give factors of 500:1 for the ratio of multipotent cellsstored per unit volume in adipose tissue relative to those stored inbone marrow.

In order to retrieve the multipotent cells from fat, a sample of fat isretrieved from the patient by techniques known in the art, generally,for example, surgery or liposuction. It has been known to utilizeenzymes, such as collagenase, or trypsin, etc., to breakdown peptidebonds in the collagen network holding the adipose tissue together, andto break down the basement membrane around the individual cells. Oncethis has been done, the multipotent cells may be separated out, andconcentrated using centrifuge, sedimentation or filtration techniques,and the concentrate is washed to remove the enzyme (residuals) used totreat the fat sample. It is thought to be vital to remove the agentsthat had been added to break down the collagen network, as these enzymesare thought to cause reduced viability of the harvested cells. Thewashed concentrate is then available for injection back into thepatient, for the purpose of accelerated repair of an injury.Unfortunately, this process to prepare a useful sample of multipotentcells, takes several hours (and in some cases up to 14 days), that makesthe ad-hoc use of such a procedure difficult or impossible, requiredmultiple processing steps, thereby increasing the potential forcontamination, compromised sterility, and the process demands skilledtechnical knowledge.

It is previously known that in addition to preparing samples ofmultipotent cells isolated from adipose tissue, the multipotent cellscould be isolated from a sample of bone marrow. However, in order toretrieve cells from bone marrow, the patient has to endure a veryuncomfortable puncture of the marrow spaces/cavities in bone (e.g., theiliac crest) before bone marrow aspirate (BMA) is drawn. The BMA sampleis then spun down in a centrifuge to gain a cellular concentrate thatcan then be injected into the patient for the repair of some injury.Although the timing of this procedure permits the ad-hoc use in anoperatory, the concentrate obtained may have an insufficient dose levelfor some applications without adopting a culturing method to increasethe concentration. The procedure utilizing BMA may be competitive toprocedures using multipotent cells from fat, however, the harvesting oftissue for BMA procedures has the disadvantage of requiring a painfulaccess procedure.

Accordingly, a need exists for a rapid multipotent cell collection,isolation and concentration apparatus and procedure that enables thead-hoc use of harvested cells in a surgical procedure, where theharvested cells can be prepared in a short timeframe (less than 5minutes), and capable of being performed following a simple protocolwith easy steps that do not require extensive technical training. Thesubject invention addresses that need (and others) by providing acompact, sterile, self-contained, easy-to-use centrifugal separationunit to provide quick and reliable multipotent cell isolation fromcollected or harvested fatty tissue and methods for quickly and reliablyisolating multipotent cells from collected or harvested fatty tissue.The fatty tissue can be collected or harvested by any means known in theart, including, but not limited to, liposuction and surgically harvestedfat. In the case of adipose tissue, the biologic mixture consists of thefatty and fibrous tissue, plus a portion of the tumescent fluids used tostabilize the fat for extraction (e.g., saline, epinephrine, lidocaine,etc.), with the multipotent cells residing in the fatty and fibroustissue. To isolate the multipotent cells for harvesting, the devicemechanically breaks down the collagen structure, and separates itsfractions by specific gravity, in order to isolate the fractioncontaining the multipotent cells for collection and use in various typesof procedures, be they diagnostic, therapeutic, or surgical.

With regard to fat processing for reimplantation, one may alternativelyobtain a sample of harvested fat to be utilized surgically, in a mannerthat does not require separating out the multipotent stem cells from thetissue structure, as described immediately above. Fat transfer, forexample, also referred to as autologous fat grafting, involves theremoval and re-implantation of a patient's adipose tissue. The adiposematerial is typically removed from areas of the body like the abdomen,thighs, or buttocks. Depending on the extraction technique (e.g.,surgical removal, liposuction, etc.), it may be necessary to remove thecertain portions of the harvested sample (e.g., tumescent solution) fromthe tissue extract. It may further be necessary, depending on thetechniques used to harvest the sample, to size the tissue, in order tocreate a homogenous product and present a material with appropriateparticulate sizes for the purpose intended. Sizing of the tissue isdesirable in many clinical applications where there is limited accessfor re-implanting the sample. For example, where there are aestheticconcerns (e.g., facial cosmetic procedures), in order to minimizescarring from incisions, the procedure may be performed by injecting thematerial via a small diameter needle. When used as a facial filler, fatgrafting can improve the creased and sunken areas of the face, and addfullness to the lips and cheeks. Fat grafting is also commonly used inbreast and buttocks augmentation, usually in place of implants.

Current fat grafting is performed by harvesting the adipose material,using a variety of techniques and surgical tools. Consequently, theproduct that is harvested may be quite different in cell viability,texture (e.g., particle size) and composition (e.g., fatty tissue,blood, tumescent solution, oil, saline, water), as a result of thetechnique utilized for harvesting. This results in variability in thematerial that may beneficially be accounted for during the processing ofthe fat sample prior to re-implantation. Furthermore, the preparationtechniques and instruments applied to the fat sample for re-implantationmay also vary, potentially resulting in a product prepared forre-implanting that may be sized to a particle size that is too small forthe intended use of the material, resulting lower cellular viabilityattributable to the excessive processing, increasing the potential forwashout of the implanted material and/or volume loss in the implantedsite. Alternatively, a sample that is sized to particle size that is toolarge for the intended use may result in challenges upon implantation,such as uneven texture, blockages of the narrow gauge needles utilizedfor re-implantation, and difficulty in the revascularization of thelarge particle size graft which may negatively affect viability.

What is needed is a device that is able to size the material to a usefulconsistency, and is able to provide a reliable composition of thematerial for implantation, regardless of the original collectiontechnique, in order to avoid the above mentioned problems.

What is needed further needed is a unitary device that can quicklyprocess, in a sterile, closed system, the fat harvested for fatgrafting, into a homogenous material, having a reliably uniform particlesize. The ideal device would consistently size the material in a mannerthat is independent of the manner of initial harvesting of the fatsample. Additionally, what is needed is a device capable of removing atleast a substantial portion of unwanted components from the harvestedsample, and preserving the components to be implanted, such as byremoving from the sample one or more of: blood, water, saline, oil,tumescent solution. Additionally, the ideal device would minimize thepotential for damage to the cellular components and tissue structurewithin the sample, in order to maximize the viability of cells to beimplanted.

SUMMARY OF THE INVENTION

In accordance with an aspect of this invention, a centrifuge forprocessing a biologic mixture, e.g., adipose tissue, and selectivelyconcentrating its constituents is provided. Those constituents havediffering specific gravities and are stratifiable in a centrifugal fieldproduced by the centrifuge. The centrifuge comprises a processingassembly and a rotation source. The processing assembly comprises aninner chamber, an outer chamber, at least one cutting element and anannular screen. The inner chamber is arranged to contain a biologicmixture, and has a central longitudinal axis about which the innerchamber is arranged to be rotated and comprises a conical member, a baseand at least one extrusion hole at a first location along the centrallongitudinal axis and extending radially through the inner chamber. Theouter chamber is arranged to receive a biologic mixture from the innerchamber and is arranged coaxially upon the central longitudinal axis ofthe inner chamber and around the inner chamber. The outer chamber isarranged to rotate about the central longitudinal axis and comprises anouter chamber wall and a dish. The at least one cutting element ispositioned between a portion of the inner chamber and the outer chamberand is arranged to remain stationary relative to the rotation of theinner and outer chambers. The annular screen is positioned between thecutting element and the outer chamber. The screen provides a series ofopenings therein and is arranged to rotate about the centrallongitudinal axis. The rotation source is coupled to the inner and outerchambers.

In accordance with another aspect of this invention a centrifuge forselectively concentrating at least one constituent of a biologicmixture, e.g., adipose tissue, is provided. The constituents havediffering specific gravities and are stratifiable in a centrifugal fieldproduced by the centrifuge. The centrifuge comprises an inner chamberarranged to receive the biologic mixture and has a central longitudinalaxis about which the inner chamber is arranged to be rotated. The innerchamber comprises a sidewall having a tapered inner surface, a base, anannular screen, and optionally, a trap and at least one roller. Ifpresent, the trap is located in the inner chamber adjacent the innersurface of the sidewall. The annular screen has an inner surface and islocated at a first radial distance from the central longitudinal axis.The annular screen projects away from the base. The at least one rolleris arranged to effectively roll around the inner surface of the screento propel at least a portion of the biologic mixture through the screenand away from the central longitudinal axis and towards the taperedsidewall.

In accordance with another aspect of this invention a method of forprocessing a biologic mixture, e.g., adipose tissue, and selectivelyconcentrating constituents of the biologic mixture is provided. Theconstituents have differing specific gravities and are stratifiable in acentrifugal field. The method basically entails providing the biologicmixture into an inner chamber of a centrifuge. The inner chamber has atleast one extrusion hole. The centrifuge additionally comprises an outerchamber disposed about the inner chamber. The inner chamber is rotatedabout an axis to extrude a portion of the biologic mixture through theextrusion hole. Portions of the biologic mixture from the extrusion holeare cut off to produce a morselized biologic mixture. The morselizedbiologic mixture is introduced into the outer chamber and the outerchamber is rotated about an axis to cause the morselized biologicmixture to stratify in the outer chamber into at least two concentricstratified constituent layers (e.g., one of which being multipotentcells).

In accordance with another aspect of this invention a method of forprocessing a biologic mixture, e.g., adipose tissue, and selectivelyconcentrating constituents of the biologic mixture is provided. Theconstituents have differing specific gravities and are stratifiable in acentrifugal field. The method basically entails providing the biologicmixture into an inner chamber of a centrifuge, and while rotating thechamber about a longitudinal axis, causing at least a portion of thebiologic mixture in the chamber to be sized by passing through arotating screen element having small openings therein. Continuedrotation of the chamber will cause the sized biologic mixture tostratify in the outer chamber into at least two concentric stratifiedconstituent layers.

In the various exemplary embodiments described herein, there is provideda motor or drive unit, which serves as a rotation source for theprocessing unit. Preferably, the motor unit is separable from theprocessing unit, such that the motor unit may be reused, while theprocessing unit is preferably a single-use component, though it iscontemplated that the processing unit may be cleaned and sterilized,such that it may be reused as well. The processing unit is an assembly,made up of an inner chamber and an outer chamber. The inner chamber isconstructed of a sidewall and a base. The sidewall has a tapered innersurface. The inner chamber includes one or more extrusion holesextending radially through the sidewall of the inner chamber at itswidest diameter. The inner and outer chambers are arranged to rotate andbe driven by the rotation source.

In some of the exemplary embodiments described herein, there may be astatic element positioned between the rotating inner and outer chambers.The static element has at least one cutting element which, incooperation with the one or more extrusion holes of the rotating innerchamber, serves to morselize the tissue into smaller fragments. In theseembodiments, as the inner chamber is rotated, the centrifugal forcedrives the biologic mixture through an extrusion hole, and uponencountering the cutting element of the static element, the ejectedmaterial is cut into smaller units, becoming morselized. Furthermore,some of these embodiments may also have a screen arranged between thestatic element and the outer chamber. As the morselized tissueencounters the screen, continued centrifugal force will urge thematerial through the screen, thereby capturing the fibrous material onthe screen, and passing the non-fibrous material to the outer chamber.This screen may also serve to further reduce the particle size of thematerial as it passes through the openings.

Once the morselized material is in the outer rotating chamber, thelarger diameter of the outer chamber will subject the morselizedmaterial to greater centrifugal forces, relative to those in the innerchamber, if the rotational speed is kept constant. Alternatively, shouldone want to maintain the level of G forces at a constant level, the rateof rotation could be reduced once the majority of the tissue material isin the outer chamber. While in the rotating outer chamber, themorselized material will stratify into annular layers, based upon thespecific gravity of the constituents of the biologic mixture. It isunderstood that the rotation rate may be varied during the processingand separation, such as rotating at a first velocity while the materialis within the inner chamber and while passing through the extrusion holeand past the static cutting element; then rotating at a second velocitywhile the material is within the outer chamber in order to achieve theseparation of the constituents by their specific gravities.

In various other exemplary embodiments of the device, the processingunit is an inner chamber, with an internal screen element. The biologicmixture is added to the interior of the chamber, and as the device isrotated, the material will encounter the screen. Continued rotation willurge the material through the screen, which will morselize the materialas it passes through the opening. Furthermore, the screen may capturemuch of the fibrous elements in the material, and passing thenon-fibrous elements through the openings to the chamber wall, where themorselized material can separate by specific gravity. In some of theseexemplary embodiments having a screen, an optional roller may beprovided to further urge the material through the screen. In such anembodiment, as the material spreads out along the inside surface of therotating screen, the material will encounter a roller arranged parallelto the screen, essentially rolling in place against the rotating screen,thus the material will be pushed through the openings in the screen asthe material encounters the roller.

In various exemplary embodiments described herein, the chamber wall, andthe base of the inner chamber may form a trap in order to capture thehighest density fraction of the fluid in the chamber, as theconstituents are separated by specific gravity due to the rotation ofthe centrifuge about the central longitudinal axis. This trap isarranged so that upon cessation of rotation of the chambers of thecentrifuge device, the effects of gravity overcome the centrifugal forceacting on the material within the device, the constituent fractionwithin the trap will remain within the trap, and not mix with theremaining material within the chamber, as that lighter fraction poolsdue to gravity in the center of the inner chamber. The fractionremaining within the trap may then be harvested by various techniquesand applied to tissue to aid in repair.

Alternatively, in other exemplary embodiments where the cells are beingretained within the native structure of the tissue material, asubstantial portion of the liquids will be removed from the tissue andaccumulate in the trap, however, a substantial portion of the desiredcells will remain within the inner chamber in fat for collection and usein surgical procedures where a scaffold material may be useful.

In accordance with another aspect of this invention, a centrifuge forprocessing a biologic mixture, e.g., adipose tissue, by sizing thematerial, and selectively concentrating its constituents is provided.The centrifuge comprises a processing assembly, and a rotation source.The processing assembly comprises a rotatable chamber arranged toreceive the biologic mixture, and a rotatable tube housing a rotatablesizing helix therein. The rotatable chamber comprises a sidewall with atapered inner surface, and optionally, a trap. The rotatable chamber andthe rotatable sizing helix are arranged to be driven by the rotationsource. As the sample material is introduced into the chamber throughthe delivery tube, the rotation of the helix will reduce the particlesize of the material. The chamber may be rotated about its longitudinalaxis to separate the components of the biologic mixture by specificgravity.

The isolated fraction containing the multipotent cells may be harvestedand stored for later use, or immediately directed into a patient fortreatment in a medical procedure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is across sectional views of one portion, i.e., a processing unitcomprising an inner and outer chamber, of one exemplary embodiment of acentrifuge device constructed in accordance with this invention andarranged for morselization and separation of tissue.

FIG. 2 is a cross section view of an alternate exemplary embodiment ofthe processing unit shown in FIG. 1 , wherein the inner chamber includesan inflection, and also showing the base unit making up the centrifuge.

FIG. 3 is a cross section view of an alternate embodiment of theprocessing unit of FIG. 1 additionally comprising a screen elementbetween the inner chamber and outer chamber.

FIG. 4 is a cross section view of another alternative embodiment of aprocessing unit of a centrifuge constructed in accordance with thisinvention, wherein the processing unit includes a screen element and aroller element.

FIG. 5 is an enlarged cross section view of still another alternateembodiment of a processing unit of a centrifuge with a screen elementand roller element constructed in accordance with this invention.

FIGS. 6A and 6B are respective enlarged cross sectional views of thescreen and suspended roller elements constructed in accordance with thisinvention.

FIG. 7 is a cross section view of still another alternate embodiment ofa centrifuge constructed in accordance with this invention making use ofa screen element, a roller element, and a secondary screen element.

FIG. 8 . is a cross-section view of still another alternate embodimentof a centrifuge constructed in accordance with this invention making useof an annular element, and a roller element, with one or more presentingan irregular topography.

FIG. 9A is a cross section view of another alternative embodiment of aprocessing unit of a centrifuge constructed in accordance with thisinvention, wherein the processing unit includes a delivery tube androtatable sizing helix.

FIG. 9B is an enlarged cross-section view of the embodiment of FIG. 9A,depicting the end of the sizing helix located within the processingunit.

FIG. 10 is a cross section view of another alternative embodiment of aprocessing unit of a centrifuge constructed in accordance with thisinvention, wherein the processing unit includes a screen element, adelivery tube and rotatable sizing helix.

FIG. 11 is an enlarged cross section view of still another alternateembodiment of a processing unit of a centrifuge with a screen elementand roller element constructed in accordance with this invention.

FIG. 12 is a cross-section view of still another alternative embodimentof a processing unit of a centrifuge constructed in accordance with thisinvention, wherein the processing unit includes a screen element, adelivery tube and rotatable sizing helix.

FIG. 13 is a cross section view of still another alternative embodimentof a processing unit of a centrifuge constructed in accordance with thisinvention, wherein the processing unit includes a delivery tube androtatable sizing helix.

DETAILED DESCRIPTION

Referring now to the various figures of the drawing, wherein likereference characters refer to like parts, there is shown in FIG. 1 oneexemplary embodiment of a portion of a centrifuge constructed inaccordance with this invention. The centrifuge basically comprises aprocessing unit or assembly (one exemplary embodiment 100A of which isshown in FIG. 1 ) and a base or drive unit 20 (shown in FIG. 2 ). Thedetails of the construction and operation of the processing unit 100Aand the base unit 20 will be described later. In addition, the detailsof other exemplary processing units will also be described later. Insome of the exemplary embodiments described herein, the processing unitincludes a rotatable outer chamber 102 and a rotatable inner chamber103. The inner chamber is arranged to receive a biologic mixture, suchas fibrous tissue, e.g., adipose (fatty) tissue, and to be rotated withrespect to a stationary cutting element (to be described later) toextrude the tissue past the cutting element where it is broken downmechanically and from whence the broken down tissue is introduced intothe outer chamber. The outer chamber is also arranged to be rotated toeffect the separation of the broken-down tissue components by thecentrifugal force produced by the rotation of that chamber.

While it has previously been known that the fibrous network in fattytissue can be broken down by using enzymatic agents, it is currentlysought to break down the fibrous network in the harvested adipose tissueby using solely mechanical means, so as to allow, in some embodiments,the release of the multipotent cells contained within the fibrousnetwork. This mechanical breaking down of the fibrous network shouldavoid the need to wash out an enzymatic agent, and may be accomplishedusing the various embodiments of the centrifuge devices describedherein. For clarity, the term morselized is used to describe the processof mechanically reducing a tissue having an initial fragment size intofragments of a smaller size by the centrifuges of this invention, alsoknown as sizing of the tissue. The terms “morselize” and “size” are usedinterchangeable herein.

The exemplary processing assembly or unit 100A of FIG. 1 , like theother processing units to be described later, is arranged to bereleasably mounted on the base 20. Once mounted on the base, thecentrifuge can be operated to rotate the processing unit at a high rateof speed (to be described later) about a central, longitudinal axis 125of the processing unit. The means for effecting that rotation basicallycomprises a motor 25 housed in the base unit 20 (FIG. 2 ). Theprocessing assembly is rotated upon activation of the motor 25 through acoupling 126. The coupling is preferably in the form of pair of keyedcomponents that releasably mate together, such that the base unit andthe processing assembly can be selectively engaged. While the variousprocessing units and the base unit are shown horizontal in the figuresof the drawing, it should be pointed out that in use the centrifuge isoriented so that the axis of rotation of the chambers is vertical, withthe base unit disposed on some surface and supporting the processingunit above it.

It is preferred that the base unit 20 be reusable so that it can be usedconsecutively with multiple processing assemblies. It is, however,contemplated that the base unit can be disposable, if desired. Theprocessing unit is, however, preferably disposable, but that is notmandatory providing that it can be sufficiently cleaned and sanitizedfor reuse. In the embodiment where the drive unit is reusable, the costfor the user can be kept lower than would be the case where the driveunit is disposed along with the rotatable separation unit. It iscontemplated that the act of joining of the engageable components (i.e.,the drive unit 20 and processing unit 100A) may trigger an automaticstart-up reaction in the drive unit, in order to begin processing of thefibrous material. For example, by incorporating magnetic switches in thedrive unit, the act of inserting the processing unit into the drive unitmay wake up and optionally start the drive unit. Alternatively, thedrive unit may include manually operated controls, to allow the operatorto have complete control over some or all of the processing steps.

The processing unit 100A also includes an outer housing 101 in which theouter chamber 102, the inner chamber 103 and a stationary sleeve 117 aredisposed. The inner chamber 103, stationary sleeve 117 and the outerchamber 103 will be described in detail later. The inner chamber is ahollow, tapered (e.g., conically shaped) member having a sidewall and abase. The outer chamber 103 is arranged to have the tissue to beprocessed introduced into its interior via an injection port 110. Tothat end, the inner chamber is arranged to be rotated about the centralaxis 125 whereupon the centrifugal force produced by the rotation causesthe introduced tissue to be extruded through one or more extrusion holes114 in the inner chamber. The stationary sleeve 117 is disposed betweenthe inner chamber 103 and the outer chamber 102 and includes at leastone outlet hole 115, which is arranged to receive the tissue extrudedthrough the extrusion hole(s) 114 as each is brought into alignment withthe outlet hole as the inner chamber rotates with respect to the sleeve117. This action serves to cut or otherwise shear off the tissueextruded through the extrusion hole, thereby morselizing that tissue.The morselized tissue then enters into the interior of the outer chamber102 as a slurry. The outer chamber is a hollow, tapered (e.g., conicallyshaped) member having a sidewall and a dish. As mentioned above, theouter chamber is also arranged to rotate about the central axis 125 bythe operation of the motor of the base unit. That action causes theslurry material to stratify, with the higher specific gravity migratingaway from the central longitudinal axis. The outer chamber includes anannular trap 136 located at the furthest radial distance from thecentral longitudinal axis. The trap is arranged to receive the portionof the slurry having the highest specific gravity, e.g., theconcentration of multipotent cells when the centrifuge is used toprocess adipose tissue to enable those cells to be readily recoveredfrom the trap, as will be described later.

The inner chamber 103 basically comprises a base 118 and a conicalmember 134, both being driven via a shaft 129, that is integrallyfastened to the base 118. This inner revolving assembly is mounted in asleeve bearing 119 and a large bearing 104. A stationery sleeve 117, andthe sleeve extension 124 is placed around the inner rotating base 118,with the clearance between the sleeve 117 larger end and the base 118set to a precise value, typically the tolerance is set in the range of0.001 inch to 0.02 inch, and preferably 0.001 inch to 0.005 inch. Theouter chamber 102 is mounted over the inner chamber 103 and is securedthereto at an upper joint 135. The outer chamber basically comprises adish 120 secured to the sidewall of the outer chamber at lower joint130. The dish 120 thus forms the larger end of the outer chamber, and issupported for rotation on a dish end bearing 121. The extension 124 ofthe sleeve 117 is pressed fit into a bottom plate 123, which isstationarily mounted with respect to the housing 101. Thus, in thisembodiment, all three of components 123, 124 and 117 are stationary, inthat they do not rotate when the centrifuge is activated. At least oneextrusion hole 114 is provided in the base 118 of the inner chamber 103.The extrusion hole may be formed by inserting (e.g., pressing) a smallplug 113 into a hole in the wall of the base 118, with the plug havingan extrusion hole (or extrusion nozzle) 114 on its centerline and with alead or chamfer 116 formed on the inner end of the hole. Although, onlyone plug is shown, it is contemplated that more than one plug may beprovided, such as by being distributed at intervals around thecircumference of the base 118. Alternatively, the opening of theextrusion hole 114 may be integrally formed in the sidewall of the innerchamber, e.g., the sidewall of base 118, rather than requiring adistinct plug or multiple plugs to be inserted into the opening(s). Theentrance chamfer 116 can be of any angle or can be a radius, so as toprevent fiber agglomeration at the entrance chamfer. The extrusion hole114 is shown adjacent to a conical outlet hole 115 in the sleeve 117.One or more outlet holes 115 may be provided in the sleeve 117, and asshown in cross-section in FIG. 1 , two outlet holes 115 are depicted.Typically, more than one outlet hole 115 is usually used, often six, butany desired number can be used. By varying the spacing between theprovided outlet holes, the size of the particle of fatty tissue that isejected (extruded) through the outlet holes 115 can be controlled, for agiven rotational speed of the centrifuge. As the chamber 103 is rotatedadjacent to the static sleeve 117, the conical outlet hole 115 in sleeve117, as shown in FIG. 1 , acts as a blade to sever portions of materialexiting through the extrusion hole 114, and in this manner, serves tobreak down the collagen fiber network in the starting material, to forma morselized material. It is contemplated that any kind of openinghaving a sharp edge could be used, such as a square hole, oralternatively, a knife blade mounted along one side of a round hole. Theconical outlet angle of the outlet hole 115, as shown, is depicted asaround a 60 degree included angle, but other angles can also be used.

At the small diameter end of inner chamber 103 a spring 108, a steppedwasher 107 and an end-cap 106 are located. The end cap includes threads111 and is arranged to be threadedly secured on opposing counter-threadsprovided on the upper neck 105 of the outer housing 101. These engagingthreads allow the end cap 106 to be rotated, thus providing forcompression of the spring 108, which when compressed, serves to preloadthe large bearing 104 via the stepped washer 107. The preload istransmitted via the inner chamber 103 to a sleeve bearing 119. Thesleeve bearing 119 is located between the base 118 of the inner chamber103 and the stationary sleeve 117. Thus, the preload is provided to thesleeve extension 124, from thence to the plate 123 and from thence tothe outer housing 101. A small bearing 109 is mounted in the smalldiameter end of revolving inner chamber 103 in order to allow thepassage of a non-rotating needle or cannula (or other tubular member)into the revolving chamber through the injection port 110, as the innerchamber 103 is rotating.

Although the sleeve 117 has been described as stationary ornon-rotating, it is contemplated that in an alternative embodiment thesleeve may also rotate. However, in such a case there must be differencein the rotation rates of the inner chamber and the sleeve. Inparticular, in order to achieve the goal of severing portions ofmaterial exiting through the extrusion hole 114 to form the morselizedmaterial, there need be some difference between the rate of rotation ofthe inner chamber and that of a rotating sleeve. The rotation of thesleeve may be either in the same, or opposite, direction of rotation asthat of the inner chamber. For this embodiment, so long as there ismomentary alignment of the extrusion hole 114 and the conical outlethole 115 of the sleeve, then the exiting (extruded) material may besevered into smaller particles (morselized).

In operation of the various exemplary embodiments described herein,adipose tissue can be obtained from a patient by known techniques,including liposuction or surgical excision. In the case of tissueobtained by liposuction, the fatty tissue and tumescent solution mixtureare likely to be in about a 1:1 ratio and will have passed throughsuction cannula orifices that will have reduced the fat fragments to asize of about 2 mm. This biologic mixture can be fed straight into thevarious embodiments of a centrifuge device described herein, via theinjection port 110 or stationary tube 235, as appropriate.Alternatively, In the case of fatty tissue obtained via surgicallyexcision, the fat will typically be removed from the patient as asemi-coherent mass, in contrast to the tissue collected as particlesthrough liposuction. In the case of surgically excised fat, the fatshould be broken up into smaller pieces, and then is to be mixed withportions of liquid, typically with saline or tumescent solution, up totwo times the volume of fat, though it is contemplated that otherproportions may be suitable as well. The mixing of the harvested fat andmixing liquid may be performed by passing the mixture to and fro betweensyringes having nozzles of about 2 mm before placing in the centrifugedevice.

In operation of any of the various exemplary embodiments describedherein, the adipose tissue harvested may optionally be treated with anadditive, such as a biologically active agent. It is contemplated thatone may wish treat the adipose tissue with, for example, drugs,antibiotics, cellular modifiers, pH modifiers, enzymes, blood products(e.g., whole blood, platelet rich plasma (PRP), red blood cells,platelet poor plasma (PPP), bone marrow aspirate (BMA) or bone marrowaspirate concentrate (BMAC)), prior to, or during the processing of theadipose tissue in the various exemplary embodiments described herein.Alternatively, one or more preservatives or anti-coagulants (e.g.heparin, coumarin, ethylene diamine tetra acetic acid (EDTA), citrates(e.g., Anticoagulant Citrate Dextrose A (ACDA), oxalate) may be addedalone, or other additives, to the adipose tissue prior to, or as it isbeing processed in the various exemplary embodiments of the devicesdescribed herein. It is contemplated that additives may beneficially aidin separation of cells during centrifugation, may alter the behavior ofthe cells in the harvested sample for processing as described herein, orenable the storage of the harvested tissue sample for subsequentprocessing as described herein. For example, the addition of ACDA mayprevent coagulation, allowing storage of the solution containing redblood cells or platelets, or additionally, the ACDA may serve to alterthe morphology of stem cells and platelet cells. For example, Applicantsbelieve that adding ACDA to the charge of biologic mixture may bebeneficial, in the case of platelet cells typically having a plate-likemorphology, may convert to a more spherical morphology, therebybeneficially affecting the ability of the platelet cells to separate byspecific gravity, as the more spherical shape of the cell may maneuvermore easily through the other constituents of the biologic mixture,e.g., adipose tissue particles.

As mentioned earlier, the centrifuge device of FIG. 1 is mounted withits spinning axis, i.e., central longitudinal axis 125, orientedvertically. The rotating chambers are driven by the drive unit via acoupling 126 to about 15,000 rpm, or the equivalent of 4000×G at theinner chamber 103 periphery. The charge of fatty tissue and solution mixis injected into the top of the rotating device (through small bearing109) into the inner chamber, using a syringe with a narrow cannula. Asthe charge is rotating within the inner chamber 103, the charge willthen generate pressure from the centrifugal effects and attempt toextrude from the extrusion hole 114. When the extrusion holes 114 of thebase are in radial alignment with outlet holes 115 of the sleeve, asshown in FIG. 1 , the fatty tissue charge can extrude from the extrusionholes 114. However, as the inner chamber 103 revolves, the hole 114moves into a closed area where the sleeve 117 has no hole. The period oftime that the extrusion hole 114 is open is controlled by the speed ofrotation and the size of the extrusion holes 114 and sleeve outlet holes115. The extrusion flow rate of the charge is controlled by thepressure, which is in turn derived from the speed of rotation whichcreates the centrifugal field. By selection of hole sizes and rotationspeed, the length of extruded charge cut off can be determined. Withextrusion holes 114 of about 1.7 mm in diameter, sleeve outlet holes 115of about 3 mm diameter, and a chamber 103 rotation speed ofapproximately 15,000 rpm, the extruded fragments can be cut into lessthan 1 mm lengths and appear as a slurry of morselized fatty material.In an embodiment having 6 outlet holes 115 in the sleeve, a charge of 30mL passes through the extrusion holes 114 in about 20 seconds. Once thecharge has been morselized, and has passed into the outer chamber 102 asa slurry, the rotational speed may then be reduced to approximately10,000 rpm (equivalent to 3500×G for a 60 mm outer chamber diameter),and the centrifugation continued for an adequate period of time, e.g.,approximately 2 minutes, to ensure separation of the desiredconstituents.

During this centrifugation process, the fatty constituent of thematerial tends to migrate toward the central longitudinal axis 125, andthe heavier cells and aqueous solution tend to move toward the outerwalls of the outer chamber 102. The heaviest density fluid (having thehighest specific gravity), containing the highest concentrations ofmultipotent cells, moves to the outermost diameter, to the annular trap136. That trap basically comprises an angularly extending channel,though the size and shape of the trap may be modified to capturedifferent fractions of the biologic mixture. For example, the trap maynot be angled as shown, but rather may be a channel that is arrangedparallel to the axis of rotation 125. In any event, when the rotation ofthe centrifuge chambers is stopped, the fraction of the biologic mixturenot within the trap 136, and within the chamber 102, then drops into acavity 151 in dish 120 by gravity, and the more viscous fat materialcollapses onto this liquid. Thus the multipotent cell containing liquidcan be isolated in the trap 136 for harvesting. The liquid containingthe majority of multipotent cells residing in trap 136 may then beremoved by syringe and a shaped cannula, via ports 138 and 137 in thehousing 101 and the outer chamber 102, respectively.

For greater ease of manufacture, the inner chamber 103 and outer chamber102 are arranged to rotate together, in a synchronous fashion, however,in this and in the other embodiments described herein, it iscontemplated that the centrifuge could be arranged so that the innerchamber 103 and outer chamber 102 rotate in an asynchronous manner.Thus, the inner chamber 103 may rotate at a first speed, so long as thatrotation creates a centrifugal field which will generate sufficientpressure upon the charge of tissue, so as to cause the ejection of thetissue material through the extrusion hole 114; while the outer chamber102 rotates at a second speed, whether in the same or differentdirection of rotation, so long as the rotation creates a centrifugalfield, so as to effect the stratification of the morselized slurrymaterial.

It has been observed that fat of different composition behavesdifferently in the centrifuge device. Whereas a portion of the liquidhaving the highest specific gravity does indeed move to the outerdiameter during centrifugation and a portion of that highest specificgravity fluid fills the trap 136, the residual fat may, or may not,emulsify into a stable creamy paste. In those instances where theresidual fat is in the form of a stable paste, the paste material willbe self-supporting, at least for a few minutes, rather than flowing, aswould a paste that is not self-supporting. If the paste is relativelystable, upon ejection through the extrusion hole 114, the paste may coatthe inner wall of the outer chamber 102 at smaller diameters, nearer thecone apex, with the paste remaining in place against the wall, evenafter the rotation of the centrifuge has been stopped. Alternatively thefat can remain as small granules, that do not adhere to either the outerchamber 102 wall or to each other, rather these granules remain free tomove relative to each other, in contrast to a material having aself-supporting paste consistency. In these instances, when the chamberstops spinning, the fat granules tend to fall toward the chamber's largediameter end and may disturb the higher specific gravity fluid that hasbeen collected in the trap 136, potentially reducing the concentrationof that fraction. To minimize the possibility of the granules of fatinterfering with the liquid collected in the trap, the centrifuge mayinclude an alternative processing unit 100B as shown in FIG. 2 . In thatembodiment the wall of the outer chamber 102 includes an off-set orinflection 141 which serves to ensure that the inner diameter 139 ofthat portion of the outer wall 102 is reduced to about that of the traplip 140. This arrangement allows the fat granules to drop into the basin151 of dish 120 and miss the trap 136.

Referring to FIG. 3 , there is shown another alternative embodiment of aprocessing unit 100C. This embodiment includes screen element 150located between the stationery sleeve 117 and the outer chamber 102. Thescreen 150 is a mesh-like element 150 and is arranged so that the slurrywhich exits from the extrusion holes 114 encounters the screen, therebymorselizing the slurry into smaller particles that pass through thescreen. The screen 150 is arranged to rotate along with the extrusionholes 114, thus the screen need not necessarily extend entirelycircumferentially around the sleeve 117. Instead it may be radiallyaligned with the exit holes 114. However, for ease of manufacture, onemay incorporate the screen 150 as a concentric ring surrounding thesleeve 117. In operation of the device, as the fatty tissue slurry exitsthe extrusion hole 114 under pressure generated by the rotation of thedevice, it impacts on the screen 150. The screen can be a wire mesh or aperforated tube, with holes of a diameter most likely in the range 1 mmto 0.25 mm, but in general of whatever size best breaks down theadipocyte binding structure. Ideally, the screen is of metal wire meshconstruction, though plastic mesh may work as well. As the screen isrevolving, any slurry of fat, fibers, and liquid will experiencesignificant centrifugal forces to propel portions of the slurry throughthe screen. Experiments have shown that the slurry is broken downcompletely, leaving only fibers on the screen, and a high count ofmononuclear, including multipotent cells in the liquid. Like theoperation discuss above, the continued rotation of the chambers of thisembodiment of the centrifuge causes the liquid components that haveflowed through the screen to separate by specific gravity in the outerchamber 102, with the highest specific gravity components accumulatingin the trap 136. As rotation is halted, the remaining fluid collects inthe basin 151. The multipotent cell rich liquid in the trap 136, maythen be harvested by directing a needle or cannula through ports 137 and138 and into the trap 136.

With this embodiment of the device, and when processing porcine deepadipose tissue, it is possible to retrieve up to 90% of the viablemononuclear cells from the fat samples.

FIG. 4 shows another alternate embodiment of the processing unit 100D.This processing unit is also arranged to be driven from a motor having adrive axis, and housed in a base unit, as has been described previously.Unlike the previously disclosed processing unit embodiments, theprocessing unit 100D of FIG. 4 includes a roller element 210, acting inconcert with a rotating screen 215 to morselize the tissue providedwithin the inner chamber, prior to causing the tissue to be separated bycentrifugation. This embodiment includes a revolving inner chamber 103having a conical sidewall 134. An annular screen element 215 is locatedwithin the inner chamber 103 and extends concentrically around thecentral longitudinal axis (i.e., the axis of rotation). The screenelement extends from where it is joined to the base 118 of the innerchamber, up to the point where it meets the chamber's sidewall. Thescreen thus divides the inner chamber 103, so that material passing froma region within the annulus of screen element 215 to the outside of thescreen element must necessarily pass through the openings provided inthe screen element. The screen element is a mesh-like member that may bea metal or polymer wire material, or alternatively a perforated sheetproviding openings sized to pass fluid material, but retain much of thefibrous material. It is envisioned that the openings will be uniformlyor non-uniformly sized between 0.002 and 0.040 inches. To aid in passingthe tissue material through the screen element, a roller element 210 isprovided adjacent to, and arranged to roll against, the inner surface ofthe screen element. The roller element is mounted on an axle 220. Theaxle may be of any sort known in the art. As shown in FIG. 4 , the axlemay be a formed stiff wire that extends through the center bore of theroller, and the wire is mounted so that it may be secured in a static(stationary) position within the inner chamber. In this embodiment, theupper end of the wire that forms an axle 220 that is fixed to a flange225 at the end of a stationary tube 235 and which extends through anopening in the end cap 106. The flange 225 and the stationary tube 235feature a hollow bore extending through their interior, which serves asthe entry port for directing tissue material into the device forprocessing. The flange 225 and stationary tube 235 are isolated from therotation of the inner chamber 103 by flange bearings 230 and a portbearing 245. The lower end of the formed wire that makes up the axle 220is directed into a bushing 240, placed in the concentric center of thebase 118 of the inner chamber 103, in line with the axis of rotation ofthe inner chamber. The bushing 240 serves to isolate the static axle 220from the rotation of the inner chamber 103. Thus, the roller element 210can effectively be made to roll around the interior surface of thescreen element 215 by keeping the roller axle 220 stationery as thescreen 215 and inner chamber 103 revolve.

It is contemplated that there may be a benefit to utilize a rollerelement 210 which is provided with a freedom of movement, such that itcan articulate, as it rotates about the static axle 220. Examples ofpossible articulation mechanisms are shown in FIGS. 6A and 6B. Byproviding a force at the mid-length of the roller, and clearance overthe axle 220, the roller can move relative to the chamber's axis ofrotation as it engages lumpy portions of the tissue material. FIGS. 6Aand 6B show a detailed expanded view of the roller element 210, on theaxle 220, against the screen element 215. In these embodiments, theroller is able to float against the screen element 215, by the nature ofthe deflection in a direction perpendicular to the axis of rotation,which is allowed by the spring wire formed as axle 220. Further, theroller element 210 is able to yaw, demonstrated as the rotation axis ofthe roller element 210 leaning, as the roller element 210 encounters thetissue against the screen element 215. The ability to yaw is provided asthe roller may pivot on the axle 220.

Referring again to FIG. 4 , it can be see that the inner chamber 103features a tapered sidewall 134. The base 118 of the inner chamber 103is shaped so as to provide a tapered surface as well, relative to theaxis of rotation 125, provided by the wedge 265. As is depicted in FIG.4 the trap 136 is in this case defined by the outer surface of the wedge265 and the inner surface of the conical sidewall 134. The trap 136 ispreferably annular, though it is contemplated that alternate shapes maysuffice, for example by being lobed. The trap, as depicted in FIG. 4 incross section, appears as an angled passageway, with an innermostportion at the entrance into the trap, where the mixture componententers from the central area of the rotating chamber, and adjacent tothe tapered end of the wedge 265. The entrance to the trap forms the endof the trap closest to the axis of rotation 125. The trap 136 alsofeatures an outermost portion, at the end having the greatest radialdistance from the axis of rotation 125. Alternatively, the trap 136 maynot be angled relative to the axis of rotation, but rather may bearranged parallel to the axis of rotation. At the largest outsidediameter of the trap, there is provided a first port 275, which isselectively openable, such as through valving, so as to allow access tothe trap to harvest the processed material fraction contained therein.An access opening 270 may be provided in the outer housing 101 tofacilitate access to the first port 275. In particular, when the firstport 275 is to be accessed, an access needle or cannula (not shown) maybe directed through aligned openings, so as to allow the harvest of theprocessed material fraction in the trap 136. This may be accomplished byplacing into alignment the access opening 270, the first port 275, andthe sealed port 271. That port may be in the form of a duckbill valve ora self-sealing septum in the wall of a container or vessel 272. Thecontainer 272 is an annular member located within the lower portion ofthe processing chamber 100D and its function will be described later.The alignment of the access opening 270, the first port 275, and thesealed port 271 may be controlled by various means known in the art, forexample, by manually rotating a portion of the exterior housing. Cams273 are provided projecting inward from the bottom portion of thehousing to selectively adjust the vertical positioning of selectedelements in the device. Alternatively, the first port may be selectivelyopenable, and closeable, during the rotation of the inner chamber 103,such that at least a portion of the material contained within the trap136 may be automatically ejected into a collection area that may beaccessed later.

In operation of the embodiment depicted in FIG. 4 , a charge of fat andsolution, such as blood, saline, water, tumescent solution, is insertedthrough the stationery tube 235. Once the charge has been inserted, theinner chamber 103 and screen element 215 are rotated via the coupling126, by the motor at a first speed, while keeping the roller axle 220stationery, for a defined first period. During this first period ofrotation, the fat will tend to spread along the inside of the screenelement due to the effect of the centrifugal field created by rotationof the chamber, and the roller element 210 will force the fat throughthe mesh of the screen element 215, as the fat passes between the roller210 and the screen element 215. The tissue material becomes morselizedinto smaller particles by being forced through the openings in the mesh,and further, a portion of the collagen fibers become separated from theother materials in the charge and are retained on the mesh by becomingdraped around the screen wires. It is also possible that uponencountering the roller and being forced through the screen, thecollagen fibers in the fat material are cut by the mesh, and thus thetissue charge is morselized into smaller particles sizes. Subsequent tothe charge being forced through the mesh, the inner chamber 103 is thenrotated at a second speed to centrifuge the morselized material, andbased on the specific gravities of the components making up themorselized material, separate the mixture of liquid and fat for a seconddefined period of rotation. During this second period, it is believedthe heavier multipotent cells tend to migrate through the liquid to theoutermost surfaces in the inner chamber. In particular, while the innerchamber 103 is rotating about the axis 125, the centrifugal fieldcreated will create stratification of the constituent components bytheir specific gravity, as the centrifugal field will urge the highestspecific gravity components away from the axis of rotation (i.e.,outwards), whereupon they will encounter the tapered walls of the innerchamber 103 and the inner surface of the wedge 265. Continued rotationwill cause these more dense components to displace less densecomponents, as the higher specific gravity components gather along thetapered sides out from axis of rotation, whereupon the highest specificgravity components will then enter into, and accumulate, in the trap136. When the rotation of the inner chamber 103 is stopped at the end ofthe second defined period, the residual liquid settles in the base 118and is collected within the dished area 250, defined by the area beingsurrounded by wedge 265 on its perimeter, and having the base 118 as abottom surface. The fat material that had been held towards the centerof the centrifugal field, due to the lower specific gravity, willfrequently have the consistency of a paste, and either tends to remainstuck to the upper portion of inner surface of the sidewall 134, oralternatively the fat material may settle within the dished area 250.The liquid containing multipotent cells remain in the trap 136, on theoutside of the wedge 265, and may be retrieved by using a needle orcannula (not shown) to suck out the fluid and cells from the trap 136.As mentioned earlier, the embodiment shown in FIG. 4 also provides afirst port 275, which may be selectively opened and closed by valving,to allow removal of higher specific gravity components from the trap136. Furthermore, there may also be provided a second port 280, whichmay be selectively opened and closed by valving, to allow removal oflower specific gravity components from the dished area 250. The secondport 280 may be located at the base of the wedge 265 within the dishedarea 250. By selectively opening the first or second port for a periodof time while the chamber is being rotated, one may fine tune thespecific gravity of the cellular concentrate fraction that is collectedwithin the inner chamber 103 in a manner similar to that described inco-pending U.S. patent application Ser. No. 13/396,600, which isassigned to the same assignee as this invention, and whose disclosure isincorporated by reference herein. The centrifuge of that application isparticularly suitable for obtaining a desired fraction from a biologicliquid mixture, such as platelet rich plasma from whole blood, or stemcells from bone marrow aspirate, however that application provides nocapacity for morselizing tissue structure in the biologic mixture, suchas adipose material.

In any of the various embodiments described herein, wherein there is achamber comprising one or more of: a wedge element 265, a first port275, or a second port 280, the ejection of one or more portions of thebiologic mixture within the chamber may be accomplished as follows. Thebiologic mixture, having been sized by any of the methods describedherein, is then rotated within the rotatable chamber to cause thecontents to separate by specific gravity. Thus, an outer band of highdensity fluid (having a higher specific gravity) will form, uponrotation of the chamber, at the outermost surface of the chamber(farthest away from the longitudinal axis 125. An inner band of lowdensity fluid (having a lower specific gravity) will form in the liquidclosest to the center of the chamber (closest to the longitudinal axis125). In between, the outermost and innermost layers, will be at leastintermediate layer comprising at least one fraction having a specificgravity between that of the innermost and outermost layers. It iscontemplated that the rotation of the chamber and its contents will forman air core, where there is no fluid at the longitudinal axis, so longas the volume of fluid in the chamber is less than the volume of thechamber itself. In those embodiments, where there is a need to eject outof the chamber the heaviest fraction of the biologic mixture, forexample, where the fraction having the highest specific gravity containsalmost no multipotent cells, this outermost fraction may be dischargedthrough selectively openable first port 275 having an inlet within thechamber at the greatest distance from the longitudinal axis, such thatwhen the valving for the first port is opened, the rotation of thechamber will create a centrifugal force urging the liquid with thehighest specific gravity to exit the chamber through the first port 275.The first port is to remain open to allow at least a portion of thehighest specific gravity fraction to exit the chamber, whereupon thefirst port may be closed, whether by action of the operator monitoringthe location of an interface, on the tapered surface of the chamber, oroperation of an automatic valve. For example, the operator may monitor acolor interface that occurs between red blood cells and the multi-potentstem cell fraction, which can be detected through a transparent sidewallof the centrifuge devices described herein. Furthermore, in thoseembodiments where there is a need to eject the lightest fraction of thebiologic mixture, for example, where the fraction having the lowestspecific gravity contains almost no multipotent cells, this innermostfraction may be discharged through selectively openable second port 280,having an inlet located within the chamber at a radial distance that isless than that of the radial distance for the inlet of the first port275, such that when the valving for the second port is opened, therotation of the chamber will create a centrifugal force urging thefraction of the liquid with the lowest specific gravity to exit thechamber through the second port 280. The second port may remain open toallow at least a portion of the lowest specific gravity fraction to exitthe chamber, whereupon the second port may be closed, whether by actionof the operator or operation of an automatic valve. In many instances,the second port may be allowed to remain open until the air core,expanding as fluid exits chamber, reaches the entrance to the secondport 280, thereby cutting off the flow of fluid out of the second port.In this manner, the inner band of lower density fluid (having a lowerspecific gravity) and optionally, fat, can be discharged through thesecond port 280 and into the container 272, leaving the desiredconcentrate fraction within the dished area 250, at the center of theinner chamber 103, once the rotation ceases. The at least one fraction,having a specific gravity intermediate that of the 2 ejected fractions,will remain within the chamber, and may then be collected by insertionof a cannula into the chamber.

FIG. 5 shows yet another alternative embodiment of a processing unit100E. That unit, while somewhat different structurally, operatessimilarly to the processing unit 100D shown in FIG. 4 , in that the unit100E includes a screen and roller arrangement that serves to morselizethe tissue material, as has been described above. In the embodiment ofFIG. 5 , a charge of tissue is delivered to the inner chamber 103, andthe inner chamber is rotated. The centrifugal field generated by therotation will cause the charge of tissue to spread along the screenelement 215, whereupon the tissue will be forced through the screenelement under the pressure of the roller 210, rotating around a rolleraxis 220. As before, the passage through the screen element morselizesthe tissue, and may retain or cut, the collagen fibers in the charge.The morselized tissue will continue to rotate with the rotation of theinner chamber, causing the stratification of the components of themorselized tissue to separate by specific gravity, with the lowestspecific gravity components being displaced at the perimeter by thehigher specific gravity components, as the higher specific gravitycomponents are driven away from the axis of rotation 125.

In this or the other processing unit embodiments having a screen element215, there may be included an optional secondary screen element 216. Insuch a case, the morselized tissue that has been directed through thescreen element 215, will encounter the secondary screen element 216, asthe material is directed outwards by the force of the rotation. Thesecondary screen element 216 is similar to the screen element 215,except that it has a smaller average opening size. While the secondaryscreen 216 may serve to further morselize the tissue, it is primarilyintended to capture the fibrous material that does not readily passthrough the openings, while passing the liquid and non-fibrous materialtherethrough. Use of this arrangement may benefit from reducing therotational velocity while the processed material is encountering thesecondary screen, so as to avoid having excessive centrifugal forcespropel the material through the screen, where a slower rotation wouldaid in capturing the fibrous material against the screen while theliquid is urged through the openings.

As should be appreciated by those skilled in the art by reference toFIG. 5 , while the rotation is ongoing, the highest specific gravitycomponents will, under the force generated by the inner chamber'srotation, accumulate in the trap 136. Upon cessation of rotation of theinner chamber 103, all of the material that is not retained within thetrap, will fall, under the influence of gravity, into a dished area 250in the center of the inner chamber. A cannula, needle or tubing may thenbe inserted through an access route created by ports 138 and 137 nearthe top of the device, optionally directed through an opening providednear the top of the optional secondary screen element 216, and directedinto the trap 136, so as to harvest the heaviest specific gravitycomponent, including the multipotent cells, while leaving thenon-desired constituents within the dished area 250.

In the various embodiments described herein, the angle of the innerchamber and wedge, relative to the axis of rotation, will affect howforcefully, and thus how quickly, the stratification of the variouscomponents will occur. For example, in an embodiment where the angle ofthe inner chamber and wedge is shallow, the separation of theconstituents will require an increased period of time of rotation, oralternatively higher rotation speeds may be required to drive theseparation. By contrast, in an embodiment where the inner chamber andwedge are at a steep angle, off the axis of rotation 125, this steepangle will tend to produce a more forceful and rapid separation of thecomponents. The angle required may be tailored to the viscosity of thefluid being processed. For example, where the charge of tissue is of ahigh viscosity, it is believed that a steep angle will allow moreeffective movement of the heaviest components through the fluid.Alternatively, where the charge of fluid is less viscous, it may bepossible to employ a shallow angle, and still achieve adequateseparation of the constituents. The goal of achieving rapid separationof the constituents is vital, as it is believed that extended durationof the exposure of living cells to elevated G forces during separationmay negatively affect the viability of the cells. Thus, it is believedthat minimizing the period of time in which the cells are rotated athigh speed will lead to better viability of the processed cellularmaterial. In practice of the various embodiments described herein, it isanticipated that the angle of the inner chamber and wedge will likely bebetween 5 degrees and 30 degrees, but angles of up to 45 or 60 may alsowork adequately.

In the various embodiments described herein, there may also be a benefitin aiding in the separation of the multipotent cells from the fibrouscollagen network in the biologic mixture, such as by adding a volume ofsaline or other fluids (e.g., blood, bone marrow aspirate, or other bodyfluids, buffered solutions, cell culture media, detergent solutions,therapeutic solutions such as antibiotic, or anti-coagulants, etc.), ashas been discussed previously. This additional fluid added to theharvested fatty tissue may serve to decrease the overall viscosity ofthe biologic mixture, which will in turn provide for more effectivemovement of the constituents of the mixture into stratified layers uponexposure to rotational forces. Additionally, the added fluid may enhancethe separation of the desired cellular concentration from the otherportions of the tissue sample. For example, the addition of whole bloodor bone marrow aspirate, when separated by density, will result in theplatelet-rich buffy coat comingling with the multipotent stem cells ofthe adipose tissue sample, as they would have similar specificgravities. The red blood cells, due to their highest specific gravity inthe combined sample, would tend to accumulate at the outside layerwithin the rotating chamber. The plasma of the whole blood will form aseparation layer between the multipotent cells and the fatty tissue. Theplatelets will likely form a layer adjacent to and/or intermingle withthe multipotent cells. Furthermore, the addition of whole blood or bonemarrow aspirate would also provide a visual indicator by color. Radialstratification would occur with layers forming, in order from theoutermost to the innermost, with the red blood cells outermost, themultipotent cells and platelets next, clear plasma next, and the fattytissue radially innermost, with the red blood cell boundary marking theedge of the fraction with the desired cellular constituents.Additionally, the addition of a liquid to the adipose tissue wouldlikely serve to dilute out the epinephrine and lidocaine that may havebeen added for the collection of the fat sample.

Furthermore, it is contemplated that there may be a benefit to thevarious embodiments described herein by providing an agitation stepafter the morselization step, wherein the centrifuge device is operatedin a manner that would impart a gentle, mixing movement to the biologicmixture, so as to ensure the cells are further separated from thefibrous network. The gentle mixing would thereby serve to avoidsubjecting the cells to the potentially harmful effects of extended highG forces to achieve separation of the cells from the fibrous network, asit is believed that extensive periods of rotation at high speed may bedetrimental to cell viability. This gentle mixing action may be achievedby random orbital movement, such as rocking off-axis, or alternatelystarting and stopping the rotation of the device, or varying the rate ofrotation of the device. For example, the device may be rotated in anoscillating manner, at low frequency (e.g., less than 10 Hz, preferablyaround 1 Hz) and subjecting the cells to low G forces, in order to freethe multipotent cells from the fatty and residual fiber network, or mixin additional fluid into the charge of tissue. The effect of the mixingmay be enhanced by including projections, such as fingers, ribs orradial fins, extending into the rotating chamber. Such projections canbe arranged as vertical elements, spiral elements, or combinationsthereof, on the surface of at least one of the wedge 265, the outersurface of the mesh of the screen element 215, the inner surface of theconical sidewall 134, and the base 118, so long as a mixing feature isextended into the dished area 250. The oscillating motion would be quitesimilar in operation to that of a conventional clothes washing machine,where the alternating start-stop, and optionally, oscillating movement,all at much lower speeds than would be required to achieve centrifugalseparation, should not result in significant reduction of cellviability, all the while providing the benefit of aiding in mechanicallydisassociating the cells from the fatty and residual fibrous materialand other constituents of the biologic mixture.

Another alternative embodiment of a processing unit 100F constructed inaccordance with this invention is shown in FIG. 7 . The centrifuge usingthat embodiment is designed to process the tissue material into smallerfragments by morselizing the tissue, by passing the material through afirst screen 215, with the aid of roller element 210, as describedpreviously. In this embodiment however, the first screen 215 isconfigured to morselize the material into smaller fragments, but not toseparate the cells from the structure of the tissue material, so as toensure that the cells remain contained within the native structure ofthe morselized tissue material. In this embodiment, the secondary screen216 is a smaller band nearer the entrance to the trap 136, and featuresopenings that allow the passage of liquid material, while retaining thecell-containing tissue material. In this manner, any exogenous fluid(e.g., saline, epinephrine, lidocaine, etc.) added for the collection ofthe tissue material can pass through the second screen 216, and collectin the trap 136, while the cells would remain in the native structure ofthe tissue material. Once the separation of liquid from tissue isaccomplished, the cell-containing tissue material may be removed fromthe interior of the inner chamber 103, such as by aspiration with acannula or other hollow luminal instrument directed through ports 138and 137. Where the cell containing tissue material has had the majorityof the fluids removed by the secondary screen 216, and as such, is notsuitable for aspiration as described above, it is contemplated that theoperator may simply open the device, using techniques which would beknown to those skilled in the art, in order to access the interior ofthe inner chamber 103, and manually collect the cell-containing tissuematerial.

As should be appreciated by those skilled in the art the embodiment ofFIG. 7 should be useful where a particular clinical application warrantsthe addition of a scaffold, such as where it is necessary to provide abulking agent to a treatment site (e.g., plastic surgery, cosmeticwrinkle reduction, etc.); or alternatively in procedures where it isdesirable to avoid washing away the harvested cells, for example inarthroscopic surgery where saline irrigation is commonly utilized, andmaintaining the delivered cells at the desired site would be beneficial.

Another alternative embodiment of a processing unit 100G constructed inaccordance with this invention is shown in FIG. 8 . The centrifuge ofthat embodiment is designed to process the tissue material into smallerfragments by morselizing the tissue, where the tissue material is passedbetween a roller element 210′ arranged to roll in place against arotating annular element 215′, in a manner as has been describedpreviously. It is envisioned that the annular element 215′ may be a meshscreen material, as previously described, or may alternatively featurean impermeable surface. Preferably, either one, or both, of the surfaceof the roller 210′, or the surface of the annular element 215′ featuresan irregular topography. This may be accomplished by providing recessedregions and protruding areas on the surface of the cylindrical roller210′. For example, by providing at least one recessed channel, andleaving protruding areas between the channels, and thus presenting asurface similar to the surface of a waffle iron. Alternatively, theirregular topography of either roller 210′ or annular element 215′ mayfeature protruding nubs or bumps, or recessed dimples. What is sought isfor the tissue, as it is squeezed between the roller element 210′ andthe annular element 215′, to experience higher degrees of disruption dueto the protruding surfaces, and thereby creating concentrated sheerforces in some of the tissue as it passes by the roller. It is believedthese sheer forces will provide a morselized slurry, in which themultipotent cells are freed from the containment of the fibrousmaterial. In these embodiments, it is envisioned that all of the tissuematerial that has been processed would then be collected and utilized ina clinical application, for example as a bulking agent delivered to atreatment site, or in procedures where it is desirable to avoid washingaway the harvested cells, as has been described previously with regardto FIG. 7 . Where the annular element 215′ is a mesh screen, themorselized tissue would be collected from the dished area 250, via theaccess pathway established through ports 137 and 138, as has beendescribed previously. However, where the annular element 215′ is anon-permeable surface, the morselized tissue would remain within theinterior of the annular element 215′ after processing, and may then becollected with a cannula or needle inserted through the stationary tube.

Another alternative embodiment of a processing unit 100H constructed inaccordance with this invention is shown in FIG. 9A. The centrifuge usingthat embodiment is designed to process the tissue material into smallerfragments by first sizing the tissue, by passing the charge of materialthrough a stationary tube 235, containing a sizing helix 305 that isrotatable around a non-rotating core wire 236. The core wire is affixedto the end of the stationary tube. Stationary tube 235, extends into therotatable chamber 103 and into the rotatable tube 315. In thisembodiment, the stationary tube 235 is temporarily fixed to end-cap 106,through techniques known to those skilled in the art. For example, asplit ring clamp may be incorporated into the collar of the end cap,where the stationary tube 235 passes through end-cap, such that theclamp may releasably secure the stationary tube relative to the end-cap.The biologic mixture, for example, a charge of fat material, may then bedirected through entry port 295, and passed through the interior of thestationary tube to exit through delivery port 320, to enter into thechamber. An optional fitting 290 (e.g. luer connector) may be providednear the top of the stationary tube, so as to securely connect thedelivery tube to the container (not shown), typically a syringe,containing the biologic mixture to be processed. The biologic mixture isintroduced via a vessel at least temporarily attached to fitting 290,for example by advancing a plunger of a syringe, propelling the biologicmixture through entry port 295 and into the interior of the stationarytube 235 while the sizing helix 305 is rotating. The edge of the entryport 295 may be sharpened to form a cutting edge, such that the rotationof the sizing helix may sever the tissue in the biologic mixture againstthe cutting edge of the entry port, and further, the rotation of thesizing helix 305 within the stationary tube 235 serves to reduce theparticle size of the biologic mixture to a desired range. In the case ofthe tissue material, the rotating action of the helix within thestationary tube serves to sever the tissue into smaller fragments tocreate a homogenous material, and further sizes the tissue to adesirable particle size, as the tissue is urged through the interior ofthe stationary tube to reach the interior of the rotatable chamber 103.The material that is processed through stationary tube 235 by the sizinghelix 305 is observed to be a homogenous slurry, having been sized to aconsistency suitable for narrow gauge needles used in cosmeticapplications, and is anticipated to be suitable for use in fat transferapplications. It is Applicants belief that the ideal particle size ofthe morselized tissue for re-implanting would lead to better viabilityof the implanted tissue as the size is such surface area:volume rationfor the particles would be conducive revascularization of the implantedtissue and further provide adequate nutrient flow to support cellulargrowth throughout the entirety of the implanted tissue.

Due to the unique methods of morselizing the tissue, as describedherein, whether by operation of the helix within the stationary tube, orby passing the tissue material through a mesh screen element, the tissuematerial that is processed is anticipated to be reduced to a suitableparticle size for re-implantation, but is not anticipated to causedamage to cellular components and the tissue structure, such as mayoccur by over-processing the tissue to a particle size that is toosmall. It is anticipated that by providing a tissue that is processed toan appropriate particle size, the material will have preserved cellularviability, while maintaining adequate tissue structure so as to not besusceptible to washout or significant volume loss once implanted.

In all of the embodiments having a sizing helix 305, it is contemplatedthat the drive unit 20, as shown in FIG. 2 , may be attached viacoupling 126, which is to effect the rotation of the rotatable chamber103. In turn, the rotating chamber, when rotating in one direction, willdrive the rotation of the sizing helix 305, when the drive unit 20 isactivated. With reference to the enlarged view of FIG. 9B, the sizinghelix 305 is coupled to the rotatable tube 315 as follows. The rotatabletube 315 is affixed to an insert 310, which is affixed to the end ofsizing helix 305 at connection 335, which may be in the form of asolder, weld, or epoxy joint, or other fixing technique known in theart. The rotation of rotatable tube 315 will drive the rotation of thesizing helix through the connection 335 depicted in FIG. 9B.

Referring back to FIG. 9A, the rotatable tube 315 is arranged to rotatein concert with the rotatable chamber 103 when rotated in one directiononly, through a one-way clutch and roller bearing 285 located betweenthe rotatable tube 315 and the upper end of the rotatable chamber 103.This one way clutch and roller bearing will lock up when rotation forceis applied in a first direction, thus transmitting the rotation forcefrom the chamber 103 to the rotatable tube 315 to drive the sizing helix305. However, when the rotatable chamber is rotated in the second(opposite) direction, the one way roller clutch and roller bearing 285will freewheel, and serves to isolate the rotation of the chamber 103from the rotatable tube 315, thus the sizing helix will then remainstationary as the rotatable chamber is rotating. As can be seen in FIG.9B, a platform bearing 325 which is located between the insert 310 andthe platform 330 will isolate stationary rotatable tube 315 only whenthe rotatable chamber 103 is rotating in the second direction. The legsof the platform 330 are attached to the base 118, and the platform thusrotates with the rotatable chamber 103. The platform 330 providesopenings between the platform legs, so as to allow fluid flow under theplatform, and may be similar to a 3-legged stool.

For all of the embodiments having a sizing helix 305, while in use, thebiologic mixture is to be introduced into the device while the chamber103, the sizing helix 305 and rotatable tube 315 are rotating in a firstdirection. The biologic mixture passes through the stationary tube 235,while the sizing helix is rotating about the core wire 236, within thestationary tube, and thus serves to whisk the biologic mixture, andsizes the biologic mixture to a desirable particle size that is smallerthan the initial average particle size of the biologic mixture, prior tobeing placed in the device. Once the entire sample of the biologicmixture to be processed is within the chamber 103, the direction ofrotation may then be reversed, thereby halting the rotation of thesizing helix 305, and the chamber 103 can then rotated to effect theseparation of the biologic mixture by specific gravity, as has beendiscussed previously.

As can be seen in the exemplary embodiment of FIG. 9A, the biologicmixture is introduced to the chamber 103 through the stationary tube235. The chamber is then rotated to separate the biologic mixture byspecific gravity, as has been described previously. In the case wherethe biologic mixture comprises at least fat tissue, blood and optionallysaline, water, tumescent solution, upon separation of the biologicmixture, the red blood cells, having the highest specific gravity wouldaccumulate at the outermost layer, while the fat, plasma and water, ifany, would accumulate at the innermost layer, having the lowest specificgravity. At least a portion of the outermost fraction, e.g. red bloodcells, may be discharged by opening the valving for first port 275, thenclosing the first port after an appropriate amount of the first fractionhave been ejected, as determined by observing the color interfacebetween the red blood cells and the fraction with the multipotent stemcells. The operator is able to monitor the location of the interfacethrough a transparent sidewall, so as to allow the operator to close thevalve as the interface nears the outlet for the red blood cells.Subsequently, at least a portion of the innermost fraction, e.g., plasmaand fat, and any water or tumescent fluid, may be ejected by opening thevalving for the second port 280, and closing the second port after theair core has reached the second port. The rotation may then be halted,whereupon the portion of the biologic mixture remaining within thechamber will pool at bottom of the chamber, and can be removed byinserting a cannula into the chamber. This fraction remaining willlargely consist of the multipotent stem cells and platelet rich plasma.

Another alternative embodiment of a processing unit 100M constructed inaccordance with this invention is shown in FIG. 10 . The embodiment inFIG. 10 is similar to that depicted in FIG. 9 , with the distinction ofproviding an annular screen element 215, located within the innerchamber 103 and extending concentrically around the central longitudinalaxis 125 (i.e., the axis of rotation). The biologic mixture may be sizedby passing through the sizing helix as described previously. However,once the sized material exits the rotatable tube 315 through deliveryport 320, it will be within an annular screen element 215. The screenelement 215 is affixed at each end to the rotatable tube 315, so thatmaterial passing from a region within the annulus of screen element 215to the outside of the screen element must necessarily pass through theopenings provided in the screen element. The screen element is amesh-like member that may be a metal or polymer wire material, oralternatively a perforated sheet providing openings sized to pass fluidmaterial, but retain much of the fibrous material. It is envisioned thatthe openings will be uniformly or non-uniformly sized between 0.002 and0.040 inches. Thus the screen element 215 may serve to further size thebiologic mixture material, and may further serve as a sieve, to capturefibrous elements from the disrupted tissue.

As can be seen in the exemplary embodiment of FIG. 10 , the biologicmixture is to be introduced to the chamber 103 by passing through thestationary tube 235 where it is sized by the sizing helix 305, aspreviously discussed. The chamber rotation may then be reversed to haltthe helix rotation, and to cause the material to pass through the screenelement 215. Subsequently, continued rotation of the chamber willseparate the biologic mixture by specific gravity, as has been describedpreviously. In the case where the biologic mixture comprises at leastfat tissue, blood and optionally saline, water, tumescent solution, uponseparation of the biologic mixture by specific gravity, the red bloodcells, having the highest specific gravity would accumulate at theoutermost layer, while the fat, plasma and water, if any, wouldaccumulate at the innermost layer, having the lowest specific gravity.At least a portion of the outermost fraction, e.g. red blood cells, maybe discharged by opening the valving for first port 275, then closingthe first port after an appropriate amount of the first fraction havebeen ejected, as determined by observing the color interface between thered blood cells and the fraction with the multipotent stem cells. Theoperator is able to monitor the location of the interface through atransparent sidewall, so as to allow the operator to close the valve asthe interface nears the outlet for the red blood cells. Subsequently, atleast a portion of the innermost fraction, e.g., plasma and fat, may beejected by opening the valving for the second port 280, and closing thesecond port after the air core has reached the second port. The rotationmay then be halted, whereupon the portion of the biologic mixtureremaining within the chamber will pool at bottom of the chamber, and canbe removed by inserting a cannula into the chamber. This fractionremaining will largely consist of the multipotent stem cells andplatelet rich plasma.

Another alternative embodiment of a processing unit 100J constructed inaccordance with this invention is shown in FIG. 11 . The embodiment inFIG. 11 is similar to that depicted in FIG. 4 , with the distinctionthat following elements from FIG. 4 are absent from FIG. 11 : wedge 265,trap 136, first port 275. Additionally, the base 118 now extendsdirectly to the inner surface of the sidewall 134, in a taper, ratherthan form a wedge element.

As can be seen in the exemplary embodiment of FIG. 11 , the biologicmixture is to be introduced to the chamber 103 and sized by passingthrough the screen element 215, as described with reference to FIG. 4 .Upon rotation of the chamber 103, the material will be urged throughscreen element 215 by roller element 210, as described previously. Theact of passing through the screen may disrupt the structure of thetissue material, so as to release the multi-potent stem cells from thestructure. Continued rotation of the chamber 103 will cause theseparation of the biologic mixture by specific gravity, as has beendescribed previously. In the case where the biologic mixture comprisesfat and water or tumescent solution, upon separation, the multipotentstem cells having a higher specific gravity than the fat or the water,will accumulate at the outermost layer within the rotating chamber. Atleast a portion of the innermost fraction may be discharged by openingthe valving for the port 280. It is contemplated that the fat and watercomponents would then be discharged from the chamber 103 through port280, until the air core encounters the entrance to port 280, and haltingthe discharge. It is contemplated that the fraction of the biologicmixture remaining within the chamber would include the multipotent stemcells, now having been concentrated by removal of fat and water from thebiologic mixture. Upon halting the rotation of the chamber, theremaining fraction will pool in the center of the chamber forcollection.

Another alternative embodiment of a processing unit 100K constructed inaccordance with this invention is shown in FIG. 12 . The embodiment inFIG. 12 is similar to that depicted in FIG. 10 , with the distinctionthat following elements from FIG. 10 are absent from FIG. 12 : wedge265, trap 136, first port 275. Additionally, the base 118 now extendsdirectly to the inner surface of the sidewall 134, in a taper, ratherthan form a wedge element.

As can be seen in the exemplary embodiment of FIG. 12 , the biologicmixture is to be introduced to the chamber 103 by passing through thestationary tube 235 where it is sized by the sizing helix 305, aspreviously discussed. The chamber rotation may then be reversed to haltthe helix rotation, and to cause the material to pass through the screenelement 215 as previously described. Subsequently, continued rotation ofthe chamber will separate the biologic mixture by specific gravity, ashas been described previously. In the case where the biologic mixturecomprises fat and water or tumescent solution, upon separation, themultipotent stem cells having a higher specific gravity than the fat orthe water, will accumulate at the outermost layer within the rotatingchamber. At least a portion of the innermost fraction may be dischargedby opening the valving for the port 280. It is contemplated that the fatand water components, having the lowest specific gravities, would thenbe discharged from the chamber 103 through port 280, until the air coreencounters the entrance to port 280, and halting the discharge. It iscontemplated that the fraction of the biologic mixture remaining withinthe chamber would include the multipotent stem cells, now having beenconcentrated by removal of fat and water from the biologic mixture. Uponhalting the rotation of the chamber, the remaining fraction will pool inthe center of the chamber for collection.

Another alternative embodiment of a processing unit 100L constructed inaccordance with this invention is shown in FIG. 13 . The embodiment inFIG. 13 is similar to that depicted in FIG. 12 , with the distinctionthat the embodiment of FIG. 13 lacks the annular screen element of FIG.12 . As with FIG. 12 , the base 118 now extends directly to the innersurface of the sidewall 134, in a taper, rather than form a wedgeelement.

As can be seen in the exemplary embodiment of FIG. 13 , the biologicmixture is to be introduced to the chamber 103 by passing through thestationary tube 235 where it is sized by the sizing helix 305, aspreviously discussed. The chamber rotation may then be reversed to haltthe helix rotation. Continued rotation of the chamber will separate thebiologic mixture by specific gravity, as has been described previously.In the case where the biologic mixture comprises fat and water ortumescent solution, upon separation, the multipotent stem cells, havinga higher specific gravity than the fat or the water, will accumulate atthe outermost layer within the rotating chamber. At least a portion ofthe innermost fraction may be discharged by opening the valving for theport 280. It is contemplated that the fat and water components, havingthe lowest specific gravities, would then be discharged from the chamber103 through port 280, until the air core encounters the entrance to port280, and halting the discharge. It is contemplated that the fraction ofthe biologic mixture remaining within the chamber would include themultipotent stem cells, now having been concentrated by removal of fatand water from the biologic mixture. Upon halting the rotation of thechamber, the remaining fraction will pool in the center of the chamberfor collection.

It should be pointed out at this juncture that any of the abovedescribed exemplary embodiments (or any other embodiments constructed inaccordance with the teachings of this invention) will produce aconcentrated cell fraction that may be usefully combined with (e.g.,hydrated into, mixed with, kneaded into, provided as a depot within, orlayered onto) a synthetic or natural scaffold or structure which may beimplanted into a treatment site of a living being. Such combining of thecell fraction with the scaffold may be accomplished in various manners,for example, by hydrating the scaffold with the cellular fraction,mixing the cell fraction with scaffold material, kneading the scaffoldmaterial and cell fraction together, providing the cell fraction as adepot contained within the scaffold material, coating the scaffold withthe cell fraction, applying the cell fraction as a layer alongside ascaffold material, sequentially adding the cell fraction to a targetsite followed by placement of a scaffold material to the target site, orvice versa. Various other procedures for combining a scaffold with acell fraction may be well known to those skilled in the art and may besuitable for use with the cell fraction created as described herein.

Moreover, while the previously described embodiments have focused on theconcentration of multi-potent cells, in any of the embodiments, it isrecognized that various cells along with, or instead of, the multipotentcells may be concentrated, which may include adipocytes, as well as thestromal vascular fraction (SVF) of cells including preadipocytes,fibroblasts, vascular endothelial cells and a variety of immune cells(e.g., adipose tissue macrophages, etc.). It is contemplated that bymanipulating the location of the outlet ports 275 and 280, the range ofspecific gravities to be collected can be controlled, such that all ofthe sample, or just a select portion of the cellular components in thesample can be isolated through the use of the various embodimentsdescribed herein.

The above described embodiments may be made available in kit form,including the centrifuge device and accessories needed for operation ofthe device, including instructions for use and packaging suitable forstorage and preserving sterility. In some instances, the kit may provideinstructions along with the centrifuge device (either as a single unit,or separable components), and optionally including accessories such asany or all of needles, syringes, cannulas, lidocaine, epinephrine,tumescent solution, liposuction kits and instructions for use.

As should be appreciated by those skilled in the art from the foregoingthe apparatus and methods of this invention can be used to provide aninjectable concentrate having a larger quantity of multipotent cellsthat is comparable or better than bone marrow concentrated aspirate ofthe same volume without requiring the need for a painful iliac crestpuncture to harvest cells therefrom. In addition, the subject inventionenables one to reduce the time of the procurement process of a usablemultipotent cell sample, to a few minutes, so as to allow the use of theequipment in the operatory ad-hoc, if so required. Further still thesubject invention eliminates the need for the use of enzymes orchemicals to be added to the sample for processing, yet which would needto be washed from the sample, prior to being injected back into thepatient. Thus, the subject invention overcomes the inefficiencies ofenzymatic treatments, which typically lead to lower cellular yields.

For any of the above described embodiments, it is contemplated tooptionally include a heating source, in order to maintain the biologicmixture at a temperature above ambient temperature. This may be usefulwhere the biologic mixture includes adipose tissue, and the increase intemperature, preferably to body temperature (37C) would serve to reducethe viscosity of the adipose tissue. In this manner, when the tissue isprocessed, cell viability may be improved as the cells, e.g.,multipotent stem cells, would be exposed to lower levels of shear stressduring processing. In contrast, where the processing is performed at alower temperature, the viscosity of the adipose tissue would increase,and potentially harming cell viability due to the increase in shearstress that would occur when processed by any of the embodimentsdescribed herein.

Thus since the inventive process and inventions disclosed herein may beembodied by additional steps or other specific forms without departingfrom the spirit of general characteristics thereof, some of which stepsand forms have been indicated, the embodiments described herein are tobe considered in all respects illustrative and not restrictive. Thescope of the invention is to be indicated by the appended claims, ratherthan the foregoing description, and all changes which come within themeaning and range of equivalency of the claims are intended to beembraced therein.

What is claimed is:
 1. A method for separating a component from a tissue material comprising the steps of: a. introducing a tissue material into a chamber of a centrifuge, said chamber having a central longitudinal axis about which said chamber is arranged to be rotated and said chamber comprising a sidewall with a tapered inner surface, an annular screen having an inner surface located at a first radial distance from said central longitudinal axis, and a trap, the trap being located in said chamber adjacent said inner surface of said sidewall at a second radial distance from said central longitudinal axis, wherein the second radial distance is greater than the first radial distance, b. rotating said chamber about the central longitudinal axis to pass at least a substantial portion of the tissue material through said annular screen, thereby morselizing the substantial portion of the tissue material and causing the tissue material to stratify in said chamber into at least two constituent layers as a function of differing specific gravities of the constituent layers, and c. collecting at least a portion of the highest specific gravity constituent layer in the trap.
 2. The method of claim 1, wherein the tissue material is adipose tissue and the highest specific gravity component comprises stem cells.
 3. The method of claim 1, wherein the trap is an annular trap.
 4. The method of claim 1, wherein the trap is angled relative to the central longitudinal axis.
 5. The method of claim 3, wherein the trap is angled relative to the central longitudinal axis.
 6. The method of claim 1, wherein the centrifuge further comprises a first port located in the trap.
 7. The method of claim 6, wherein the first port is located in the trap at the largest outside diameter of the trap.
 8. The method of claim 1, wherein the trap comprises an angled passageway having an innermost portion through which the highest specific gravity constituent layer enters the trap.
 9. The method of claim 1, wherein the chamber comprises a base and the annular screen projects away from the base, wherein the chamber further comprises a wedge present in the base, the wedge comprising an outer surface that is angled relative to the central longitudinal axis, and wherein an outer surface of the wedge and the inner surface of the sidewall define the trap.
 10. The method of claim 9, wherein the wedge comprises an angled inner surface, and wherein the chamber comprises a dished area in the base, the dished area at least partially defined by the angled inner surface of the wedge.
 11. The method of claim 10, wherein upon cessation of the rotation of the chamber, one or more constituent layers settle into the dished area of the chamber and do not enter the trap.
 12. The method of claim 10, wherein the centrifuge further comprises a second port located within the dished area.
 13. The method of claim 6, further comprising the step of opening the first port during the rotation of the chamber, thereby ejecting at least a portion of the highest specific gravity constituent layer from the trap.
 14. The method of claim 1, further comprising the step of removing at least a portion of the highest specific gravity constituent layer from the trap via a needle or cannula, wherein the portion of the tissue material so recovered comprises stem cells.
 15. The method of claim 6, wherein the centrifuge comprises an outer housing containing the chamber and the outer housing comprises an access opening for accessing material collected in the trap via the first port.
 16. The method of claim 15, further comprising the step of extracting stem cells from the trap via a syringe by inserting the syringe into the access opening and through the first port.
 17. The method of claim 1, wherein the chamber further comprises a secondary screen, the secondary screen being located at a third radial distance from the central longitudinal axis, wherein the third radial distance is greater than the first radial distance and less than the second radial distance, wherein the secondary annular screen is configured to capture fibrous material while allowing non-fibrous material and liquid to pass through.
 18. The method of claim 1, wherein the centrifuge further comprises a roller arranged to roll around the inner surface of the annular screen and urge at least a portion of the tissue material through the annular screen and away from the central longitudinal axis and towards the sidewall.
 19. The method of claim 1, wherein the centrifuge further comprises a roller arranged to roll against and around the inner surface of the annular screen and urge at least a portion of the biologic mixture through the annular screen and away from the central longitudinal axis and towards the sidewall.
 20. A method for separating a cellular component from a tissue material comprising the steps of: a. introducing a tissue material into a chamber of a centrifuge, said chamber having a central longitudinal axis about which said chamber is arranged to be rotated and said chamber comprising a sidewall with an angled inner surface, a trap in fluid communication with the interior of the chamber and located adjacent said inner surface of said sidewall at the greatest distance from the longitudinal axis within the chamber, and a screen located between the trap and the central longitudinal axis, b. rotating said chamber about the central longitudinal axis, c. causing at least a substantial portion of the tissue material to pass through said screen and stratify into at least two constituent layers as a function of differing specific gravities of the constituent layers, and d. directing at least a portion of the highest specific gravity constituent layer to the trap via contact with the sidewall, wherein the highest specific gravity constituent comprises a cellular component. 